One of the most promising approaches in the gene therapy of a large number of diseases involves the use of in vitro genetic modification of stem cells followed by transplantation and engraftment of the modified cells in a patient. Particularly promising is when the introduced stem cells display long term persistence and multi-lineage differentiation. Hematopoietic stem cells, most commonly in the form of cells enriched based on the expression of the CD34 cell surface marker, are a particularly useful cell population since they can be easily obtained and contain the long term hematopoietic stem cells (LT-HSCs), which can reconstitute the entire hematopoietic lineage after transplantation.
Various methods and compositions for targeted cleavage of genomic DNA have been described. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination at a predetermined chromosomal locus in cells from any organism. See, e.g., U.S. Pat. Nos. 8,956,828; 8,623,618; 8,034,598; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20060063231; 20080159996; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983; 20130177960 and 20150056705, the disclosures of which are incorporated by reference in their entireties for all purposes. These methods often involve the use of engineered cleavage systems to induce a double strand break (DSB) or a nick in a target DNA sequence such that repair of the break by an error-prone process such as non-homologous end joining (NHEJ) or repair using a repair template (homology directed repair or HDR) can result in the knock out of a gene or the insertion of a sequence of interest (targeted integration). The repair pathway followed (NHEJ versus HDR or both) typically depends on the presence of a repair template and the activity of several competing repair pathways.
Introduction of a double strand break in the absence of an externally supplied repair template (e.g. donor) is commonly used for the inactivation of the targeted gene via mutations introduced by the cellular NHEJ pathway. NHEJ pathways can be separated into the standard Ku-dependent pathway and an alternative Ku-independent pathway based on microhomology-mediated end joining, which takes advantage of short tracks of direct repeats near the cleavage site. The pattern of insertions and deletions (‘indels’) following gene editing via these two NHEJ pathways differ, which can result in differences in the functional consequences of the mutations, depending on the application.
In the presence of an externally supplied donor carrying stretches of homology to the sequences flanking the double strand break, homology directed gene repair (HDR), using the donor molecule, can be used to change the sequence of a single base or a small stretch of DNA (gene correction′ or ‘gene mutation’) or, on the other extreme, for the targeted insertion of an entire expression cassette or fragment thereof (gene addition′) into a pre-determined genomic location.
Cleavage can occur through the use of specific nucleases such as engineered zinc finger nucleases (ZFN), transcription-activator like effector nucleases (TALENs), or using the CRISPR/Cas system with an engineered crRNA/tracr RNA (single guide RNA′) to guide specific cleavage. Further, targeted nucleases are being developed based on the Argonaute system (e.g., from T. thermophilus, known as ‘TtAgo’, see Swarts et at (2014) Nature 507(7491): 258-261), which also may have the potential for uses in genome editing and gene therapy.
Targeted cleavage using one of the above mentioned nuclease systems can be exploited to insert a nucleic acid into a specific target location using either HDR or NHEJ-mediated processes. However, delivering both the nuclease system and the donor to the cell can be problematic. For example, delivery of a donor or a nuclease via transduction of a plasmid into the cell can be toxic to the recipient cell, especially to a cell which is a primary cell and may not be as robust as a cell from a cell line.
CD34+ stem or progenitor cells are a biologically heterogeneous set of cells characterized by their ability to self-renew and/or differentiate into the cells of the lymphoid lineage (e.g. T cells, B cells, NK cells) and myeloid lineage (e.g. monocytes, erythrocytes, eosinophils, basophils, and neutrophils). Their heterogeneous nature arises from the fact that within the CD34+ stem cell population, there are multiple subgroups reflecting the multipotency (whether lineage-committed) of a specific group. For example, CD34+ cells that are also CD38− are more primitive, immature CD34+ progenitor cell, (also referred to as long-term hematopoietic progenitors), while those that are CD34+CD38+(short-term hematopoietic progenitors) are lineage-committed (see Stella et at (1995) Hematologica 80:367-387). When this population then progresses further down the differentiation pathway, the CD34 marker is lost. CD34+ stem cells have enormous potential in clinical cell therapy. However, in part due to their heterogeneous nature, performing genetic manipulations such as gene knock-out, transgene insertion, and the like upon the cells can be difficult. Specifically, these cells are poorly transduced by conventional delivery vectors, the most primitive stem cells are sensitive to modification, there is limited HDR following induced DNA DSBs, and there is insufficient HSC maintenance in prolonged standard culture conditions. Additionally, other cells of interest (for non-limiting example only, cardiomyocytes, medium spiny neurons, primary hepatocytes, embryonic stem cells, induced pluripotent stem cells and muscle cells) can be less successfully transduced for genome editing than others.
For both autologous and allogeneic HSC transplantation therapies, ex vivo culture of cells derived from human donors is often necessary. Depending on the cell source, the fraction of CD34+HSPCs can be quite low—approximately 0.0005%, 0.01%, or 0.1% for mobilized peripheral blood (mPB), bone marrow aspirate (BM), or cord blood, respectively. The fraction of long-term repopulating true stem cells (LT-HSCs) within these CD34+ cell populations is even lower (<1%). Furthermore, for autologous HSC therapies, autologous cord blood is often not available and thus mPB or BM-derived HSPCs are required. For an HSC transplant to have long-term efficacy the cells must engraft into the bone marrow and produce all of the hematopoietic lineages necessary for proper immune and red blood cell function. During in vitro culture, LT-HSCs often do not survive, do not proliferate, or differentiate into lineage-committed progenitors that will not result in long-term engraftment. Moreover, using currently-available techniques, it is often difficult to modify the genomes of LT-HSCs. Therefore, for HSC transplantation therapies to produce long-term efficacy, maintaining or increasing the overall amount of nuclease-modified LT-HSCs in culture is imperative.
Thus, there remains a need for compositions and methods for genome engineering of CD34+ cells, including LT-HCSs, and other stem or progenitor cells of interest that increase the efficiency of gene modification and provide cells comprising these genetic modifications.